Stresscopins and their Uses

ABSTRACT

The invention provides novel nucleic acids and polypeptides, referred to herein as stresscopin 1 and stresscopin 2, which preferentially activate the CRH-R2 receptor over the R1 receptor. Stresscopins, analogs and mimetics, and related CRH-R2 agonists suppress food intake and heat-induced edema; but do not induce substantial release of ACTH. Stresscopin also finds use in the recovery phase of stress responses, as an anti-inflammatory agent, as a hypotensive agent, as a cardioprotective agent, and in the treatment of psychiatric and anxiolytic disorders. Stresscopin nucleic acid compositions find use in identifying homologous or related proteins and the DNA sequences encoding such proteins; in producing compositions that modulate the expression or function of the protein; and in studying associated physiological pathways.

Mammals respond to stress through interlinked endocrine, neuroendocrine,autonomic and behavioral pathways. Activation of the autonomic nervoussystem elicits the release of catecholamines, whereas hypothalamicsecretion of corticotropin releasing hormone (CRH) leads to pituitarysecretion of adrenocorticotrophic hormone (ACTH), which, in turn,stimulates glucocorticoid secretion by the adrenal cortex. Thesestress-responses can provide a vital short-term metabolic lift, but whentriggered inappropriately can also cause severe diseases. For example,anxiety and depression affect over 100 million patients worldwide everyyear, and depression is the leading cause of suicide, which claimsthousands of lives each year in the U.S. Anxiety is among the mostcommonly observed group of CNS disorders, which includes phobias orirrational fears, panic attacks, obsessive-compulsive disorders andother fear and tension syndromes. Another potentially deleteriousresponse to stress is hypertension, which can lead to fatal heartdisease and stroke.

The physiological response to stress is integrated through corticotropinreleasing hormone (CRH), and related factors (Shibahara et al. (1983)EMBO J. 2(5):775-779). CRH is a 41-amino acid peptide synthesized in thehypothalamus. It is a ligand for two receptors, CRH-R1 and CRH-R2.Another known ligand for the CRH receptors is urocortin, which is a 40amino acid peptide having substantial sequence similarity with the fishprotein urotensin and to CRH (Donaldson et al. (1996) Endocrinology137(5):2167-2170). These receptors have a seven-transmembrane structure,and belong to the family of G-protein coupled receptors, whose actionsare mediated through activation of adenylate cyclase. The type-1receptor is expressed in many areas of the brain, as well as in thepituitary, gonads, and skin (Chen et al. (1993) P.N.A.S. 90:8967-8971).The type-2 receptor is expressed in the brain, cardiac and skeletalmuscle, epididymis, and the gastrointestinal tract (Liaw et al. (1996)Endocrinology 137(1):72-77). In addition, there is a CRH-R2 spliceisoform found in human brain (Grammatopoulos et al. (1999) Mol.Endocrinol. 13(12):2189-2202).

Although the two receptors share 70% sequence identity, they differ intheir ligand binding affinity. CRH itself has a much higher affinity forCRH-R1, while urocortin is equally effective at binding both the R1 andthe R2 receptors. It is also believed that the receptors differ in theirphysiological role. An inverse relationship between the CRH-R1 andCRH-R2 receptor systems have been reported in an anxiety model,suggesting that CRH neuronal systems may be comprised of two separate,but interrelated, subdivisions that can be coordinately and inverselyregulated by stress, anxiety, or anxiolytic drugs.

Mice lacking CRH-R1 display markedly reduced anxiety, and fail toexhibit the normal hormonal response to stress (Smith et al. (1998)Neuron 20:1093-1102). Animals having a targeted disruption in CRH-R2have normal initiation of stress responses, but have deficiencies in themaintenance and recovery phases. For example, stress coping behaviorsassociated with de-arousal were reduced in these knock-out mice. Themice were also hypersensitive to stress, and displayed increasedanxiety-like behavior (Bale et al. (2000) Nat. Genet. 24:410-414).CRH-R2 may also mediate peripheral human dynamic effects, includingenhanced cardiac performance and reduced blood pressure, as well ascardiovascular homeostasis (Coste et al. (2000) Nat. Genet. 24:403-409).CRH-R2 signaling is essential for coping with the hypertension initiatedduring stress. Mutant mice have normal basal feeding and weight gain,but decreased food intake following food deprivation, suggesting a roleof CRH-R2 in feeding behavior and reduced gastric emptying. In addition,CRH-R2 signaling may be involved in the suppression of immune responsesassociated with stress.

There is considerable interest for clinical and research purposes in thediscovery and development of agents that act on these receptors,particularly where there can be enhanced specificity of action overexisting ligands.

SUMMARY OF THE INVENTION

Stresscopin nucleic acid compositions and their encoded polypeptides andvariants thereof are provided. Stresscopins are novel and selectiveligands for the CRH-R2 receptor, and thus find use where it is desirableto specifically induce the CRH-R2 and not the CRH-R1 response pathway.In addition to use as a therapeutic agent, stresscopins are utilized inscreening and research methods for the determination of specificanalogs, agonists, antagonists and mimetics.

Stresscopins, analogs and mimetics, and related CRH-R2 agonists suppressfood intake and heat-induced edema, but unlike CRH and urocortin,stresscopins do not induce substantial release of pituitary ACTH andadrenal glucocorticoids. Stresscopins also find use in the recoveryphase of stress responses, as an anti-inflammatory agent, as ahypotensive agent, as a cardioprotective agent, and in the treatment ofpsychiatric and anxiolytic disorders.

The invention also provides diagnostics and therapeutics comprisingstresscopin nucleic acids, their corresponding genes and gene products,antisense nucleotides, and antibodies specific for one or more epitopesof the stresscopin polypeptide. The nucleic acid compositions find usein identifying homologous or related genes; for production of theencoded protein; in producing compositions that modulate the expressionor function of its encoded protein; for gene therapy; mapping functionalregions of the protein; and in studying associated physiologicalpathways. In addition, modulation of the gene activity in vivo is usedfor prophylactic and therapeutic purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Nucleic acid sequence and amino acid sequence of stresscopin 1(SEQ ID NOS:1-2). The pre-pro region of stresscopin 1 polypeptide is 46amino acids and is lightly shaded, while the putative mature stresscopin1 peptide is highlighted darkly on the background (SEQ ID NO:3). Themethionine start site and the putative C-terminal amidation donorresidue are in bold letters.

FIG. 1B. Nucleic acid sequence and amino acid sequence of stresscopin 2(SEQ ID NOS:4-5). The pre-pro region of stresscopin 2 peptide is 96amino acids and is lightly shaded, while the putative mature stresscopin2 peptide is shaded darkly (SEQ ID NO:6).

FIG. 1C. Comparison of the mature regions of CRH-related peptides frommammals, fish, and frog. Amino acid numbering is given on the left. Theputative secondary structures of these polypeptides are indicated abovethe upper row of the alignment. CRH, urocortin, urotensin I, andsauvagine shared a similar structure with an N-terminal random coilfollowed by an extended α-helix structure. In contrast, the N-terminalsequences of human and pufferfish stresscopin peptides adopted anextended strand structure followed by a short random coil. Lightlyshaded residues are conserved in the majority of aligned sequences.Residues that are identical in peptides of each subgroup are highlightedby a dark background. The legend is h is human; m is mouse; g isgoldfish (Carassius auratus); s is sucker (Catostomus commersoni); f isleaf frog (Phyllomedusa sauvagei); and p is Fugu pufferfish (Takifugurubripes). CRH family peptides all have a stretch of 30 residues attheir C-termini and adopt an extended α-helical structure. Alignment ofthe mature peptides with elevated CRH family hormones indicated thatmature stresscopins from human and pufferfish, but not the pre-proregions show 35-38% identity to other family proteins. SEQ ID NOS:4,6-15.

FIG. 1D. Phylogenetic tree of CRH family proteins from vertebrates.Phylogenetic inference based on mature regions of CRH family proteins.Phylogenetic analysis of nine CRH family proteins from fish, frog andmammals suggests the ancient evolution of three subgroups of CRH familyproteins, with the human and pufferfish stresscopins clustered in aseparate branch.

FIG. 2A-2F. Expression of stresscopin transcripts and proteins. FIG. 2A,Expression of the stresscopin 1 (panels 1 and 2) and stresscopin 2(panels 3 and 4) transcripts in 23 different human tissues as determinedby PCR amplification. The specific cDNA bands are indicated by anarrowhead on the left. PCR products from experiments using two differentconcentrations of cDNA templates (1 ng template/reaction, panels 1 and3; 10 pg template/reaction, panels 2 and 4) are shown. PBL, peripheralblood cells. The expression of -actin transcripts in cDNA templates fromdifferent tissues are shown in panels 5 (1 ng template/reaction) and 6(10 pg template/reaction). FIG. 2B. Expression of the stresscopin 1transcript in 11 different human cardiac compartments as determined byPCR amplification using a primer pair flanking part of the C-terminalORF and the 3′-untranslated region of stresscopin 1 cDNA and 1 ng oftemplate cDNA. Lane 1, atrioventricular node; lane 2, atrioventricularseptum; lane 3, aorta; lane 4, apex of the heart; lane 5, left atrium;lane 6, right atrium; lane 7, dextra auricle; lane 8, sinistra auricle;lane 9, left ventricle; lane 10, right ventricle; lane 11, adult heart.The specific 177-bp stresscopin 1 cDNA bands are indicated by anarrowhead. FIG. 2C. Expression of the stresscopin 2 transcript in 12different human tissues of the digestive system as determined by PCRamplification using a primer pair flanking part of the ORF ofstresscopin 2 cDNA and 1 ng of template cDNAs. Lane 1, ascending colon;lane 2, descending colon; lane 3, transverse colon; lane 4, duodenum;lane 5, Ileocecum; lane 6, Ileum; lane 7, jejunum; lane 8, stomach; lane9, cecum; lane 10, rectum; lane 11, liver; lane 12, esophagus. Thespecific 237-bp stresscopin 2 cDNA bands are indicated by an arrowhead.FIGS. 2D and 2E. Stresscopin 1 expression in mouse cardiac (FIG. 2D) andrat pituitary (FIG. 2E) sections using anti-stresscopin 1 antibodyC2208. Specific signals (black particles) are indicated by arrows (leftpanel). Adjacent sections hybridized with anti-stresscopin 1 antibodiespresaturated with the antigen peptide showed minimal staining (rightpanels). A, atrium tissues; BV, blood vessel; AP, anterior pituitary;PP, posterior pituitary; IL, intermediate lobe. FIG. 2F, Stresscopin 2expression in mouse intestinal sections using anti-stresscopin 2antibody C2221 (left panel). A negative control with presaturatedantibodies is shown on the right panel. MM, muscularis mucosae; IG,intestinal glands; ME, muscularis externa.

FIG. 3A-3H. Stresscopin 1 and stresscopin 2 preferentially activate CRHR2. Hormonal stimulation of cAMP production by FIG. 3A. CRHR2-containingrat cardiac A7r5 cells, FIG. 3B. CRHR1-containing human retinoblastomaY79 cells, FIG. 3C. recombinant CRHR2, FIG. 3D. recombinant CRHR2 and,FIG. 3E. recombinant CRHR1. Cells incubated with the test peptide orvehicle were harvested at 16 h after treatment and heated to 95° C. for5 min to inactivate phosphodiesterase activity before cAMP measurement.FIG. 3F. Full-length 43-amino-acid stresscopin 1 (SCP1) and truncatedderivatives with deletion of 1-5 amino acids at the N-terminus (1 nM)stimulated cAMP production by recombinant CRH R2 (hatched bars), but notCRH R1 (blank bars). Nonamidated stresscopin 1 (SCP1-NA) showed aminimal stimulation of cAMP production as compared to the amidatedcounterpart. Data are the mean SEM (N=4). SCP1-0.1, stresscopin 1 (0.1nM); SCP1, stresscopin 1 (1 nM); SCP1(2-43), truncated stresscopin 1with the first amino acid deleted; SCP1(3-43), truncated stresscopin 1with 2-amino-acid deletion; SCP1(4-43), truncated stresscopin 1 with3-amino-acid deletion; SCP1(5-43), truncated stresscopin 1 with4-amino-acid deletion; SCP1(6-43), truncated stresscopin 1 with5-amino-acid deletion; SCP1-NA-0.1, full-length stresscopin 1 devoid ofamidation at the C-terminus (0.1 nM); SCP1-NA, full-length stresscopin 1devoid of amidation at the C-terminus (1 nM); UCN, urocortin. FIGS. 3Gand 3H. Competitive displacement by unlabeled CRH, urocortin,stresscopin 1, and stresscopin 2 of ¹²⁵I-labeled urocortin bound tomembranes of 293T cells transfected with CRHR2 (FIG. 3G) or CRHR2 (FIG.3H) cDNA. Data are mean SEM (N=3).

FIG. 4A-4E. CRH and urocortin, but not stresscopin 1 and stresscopin 2,stimulate ACTH release by cultured anterior pituitary cells in vitro(FIG. 4A) and induce ACTH secretion in vivo (FIG. 4B). ACTH contents inculture media and serum were determined using a radioimmunoassay. For invivo studies, male rats were injected i.p. with test peptides (2nmoles/kg B.W.) and sacrificed 30 min later. Data are mean SEM (N=4).FIG. 4C. Anti-edema response regulated by stresscopins. Stresscopin 1(20 nmoles/kg), stresscopin 2 (100 nmoles/kg), and related hormones (20nmoles/kg) suppress heat-induced paw edema formation in anaesthetizedrats (N=6). FIG. 4D. Cumulative food intake in mice treated withstresscopin 1 (left panel, N=6) and stresscopin 2 (right panel, N=4)peptides and other hormones at 2, 4, and 8 h after treatment. FIG. 4E.Reduction of gastric emptying by stresscopin 1 (blank bars, 8 nmoles/kg;hatched bars, 80 nmoles/kg), stresscopin 2 (blank bars, 80 nmoles/kg;hatched bars, 200 nmoles/kg), and related hormones (blank bars, 8nmoles/kg; hatched bars, 80 nmoles/kg) at 2 h after hormone treatment(N=6). The rates of gastric emptying were calculated by the formula (wetweight of stomach at 2 h after treatment/wet weight of stomach in fedanimals sacrificed at 0 h). SCP1 is stresscopin 1; SCP2 is stresscopin2; UCN is urocortin; SCP1 (11-43) is truncated stresscopin 1 with thefirst 10 amino acids deleted.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The invention provides novel nucleic acids and polypeptides, referred toherein as stresscopin 1 and stresscopin 2, which are members of thecorticotropin releasing hormone family. Because stresscopinspreferentially activate the CRH-R2 receptor over the R1 receptor,stresscopins, analogs and mimetics, and related CRH-R2 agonists suppressheat-induced edema as the result of their hypotensive actions as well asfood intake; but do not induce substantial release of ACTH. Stresscopinalso finds use in the recovery phase of stress responses, as ananti-inflammatory agent, as a cardioprotective agent, and in thetreatment of psychiatric and anxiolytic disorders.

The nucleic acid compositions of the subject invention find use inidentifying homologous or related genes; for production of the encodedprotein; in producing compositions that modulate the expression orfunction of its encoded protein; for gene therapy; mapping functionalregions of the protein; and in studying associated physiologicalpathways. In addition, modulation of the gene activity in vivo is usedfor prophylactic and therapeutic purposes. The proteins are useful as atherapeutic, as an immunogen for producing specific antibodies, inscreening for biologically active agents that act in the CRH signalingpathways and for therapeutic and prophylactic purposes.

Stresscopins are natural agonists of the CRH-R2 receptor, where the term“agonist” refers to a compound that binds to, and activates a receptor.Compounds that inhibit this effect are referred to as “antagonists.”Ligands, e.g. variants, derivatives and mimetics of stresscopins, mayevoke a spectrum of responses ranging from full CRH-R2 activation byagonists to partial activation and inhibition by partial or completeantagonists.

Stresscopin Polypeptides

The mature stresscopin 1 polypeptide is a 43 amino acid peptide, derivedfrom a 112 amino acid precursor protein. The amino acid sequence of theprecursor protein and mature protein are provided as SEQ ID NO:2 and SEQID NO:3, respectively. The nucleotide sequence of the human stresscopin1 cDNA is provided as SEQ ID NO:1. The mature stresscopin 2 is a peptideof 40 amino acids, derived from a 161 precursor protein. The amino acidsequence of the precursor protein and mature protein are provided as SEQID NO:5 and SEQ ID NO:6. The nucleotide sequence of stresscopin 2 isprovided as SEQ ID NO:4.

Both human stresscopin ORFs contain a signal peptide for secretion andthe predicted mature regions are flanked by potential proteolyticcleavage sites and an α-amidation donor residue. The identity of thestresscopin 1 and 2 transcripts was confirmed following PCR of cDNA fromhuman testis and colon, respectively.

For use in the subject methods, either of the native stresscopin forms,modifications thereof, or a combination of forms may be used. Peptidesof interest include fragments of at least about 12 contiguous aminoacids, more usually at least about 20 contiguous amino acids, and maycomprise 30 or more amino acids, up to the provided peptide, and mayextend further to comprise other sequences present in the precursorprotein.

A fragment of a stresscopin peptide may be selected to achieve aspecific purpose. For example, deletions at the amino terminus ofpeptides having binding affinity for CRH-receptors have the effect ofturning an agonist peptide into an antagonist, by retaining the receptorbinding activity, but deleting the activation activity (for example, seeRuhmann et al. (1998) P.N.A.S. USA 95:15264-15269). Such deletionsgenerally extend from residue 1 through 10 of the peptide, and mayfurther delete additionally amino acids at residues 11, 12 or more.Smaller deletions, of from 1 to to 5 amino acids, may be deleted in theN-terminus and still retain the agonist properties.

The sequence of the stresscopin polypeptide may be altered in variousways known in the art to generate targeted changes in sequence. Thepolypeptide will usually be substantially similar to the sequencesprovided herein, i.e. will differ by at least one amino acid, and maydiffer by at least two but not more than about ten amino acids. Thesequence changes may be substitutions, insertions or deletions. Scanningmutations that systematically introduce alanine, or other residues, maybe used to determine key amino acids. Conservative amino acidsubstitutions typically include substitutions within the followinggroups: (glycine, alanine); (valine, isoleucine, leucine); (asparticacid, glutamic acid); (asparagine, glutamine); (serine, threonine);(lysine, arginine); or (phenylalanine, tyrosine).

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps; e.g.by exposing the polypeptide to enzymes which affect glycosylation, suchas mammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

Also included in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques and syntheticchemistry so as to improve their resistance to proteolytic degradationor to optimize solubility properties or to render them more suitable asa therapeutic agent. For examples, the backbone of the peptide may becyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Analogs of such polypeptides include those containingresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids.

The subject peptides may be prepared by in vitro synthesis, usingconventional methods as known in the art. Various commercial syntheticapparatuses are available, for example, automated synthesizers byApplied Biosystems, Inc., Foster City, Calif., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

Compound Screening

The availability of purified stresscopin and other components in thesignaling pathways, e.g. CRH-R1, CRH-R2, etc., allows in vitroreconstruction of the pathway. Two or more of the components may becombined in vitro, and the behavior assessed in terms of activation oftranscription of specific target sequences; modification of proteincomponents, e.g. proteolytic processing, phosphorylation, methylation,etc.; ability of different protein components to bind to each other,etc. The components may be modified by sequence deletion, substitution,etc. to determine the functional role of specific residues.

Drug screening may be performed using an in vitro model, a geneticallyaltered cell or animal, or purified stresscopin protein. One canidentify ligands or substrates that compete with, modulate or mimic theaction of stresscopin. Areas of investigation include the development oftreatments for suppression of food intake; suppression of edema;enhancing the recovery phase of stress responses; as ananti-inflammatory agent; as a cardioprotective agent; in the treatmentof psychiatric and anxiolytic disorders, etc.

Drug screening identifies agents that mimic stresscopin activity, eitheras an antagonist or as an agonist. A wide variety of assays may be usedfor this purpose, including labeled in vitro protein-protein bindingassays, electrophoretic mobility shift assays, immunoassays for proteinbinding, and the like. Knowledge of the 3-dimensional structure ofstresscopin, derived from crystallization of purified syntheticstresscopin protein, leads to the rational design of small drugs thatspecifically inhibit stresscopin activity.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of altering or mimicking thephysiological function of stresscopin. Generally, a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typicallyone of these concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin, etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

For example, a number of molecules have been described as antagonists oras agonists of CRH receptors. Screening assays that utilize stresscopinpermit the improved selection for compounds having a desiredspecificity, of acting specifically on CRH-R2. Examples of such CRHagonists and antagonists include, among others, arylamino fusedpyrimidines (U.S. Pat. No. 6,107,300); thiazolo[4,5-d]pyrimidines andpyridines (U.S. Pat. No. 6,107,294); pyrazoles and pyrazolopyrimidines(U.S. Pat. No. 6,103,900); aryl- and arylamino-substituted heterocycles(U.S. Pat. No. 6,103,737); tetrahydropteridines (U.S. Pat. No.6,083,948); benzimidazole derivatives (U.S. Pat. No. 6,022,978);substituted 4-phenylaminothiazoles (U.S. Pat. No. 5,880,135);benzo(e)perimidine-4-carboxamide derivatives (U.S. Pat. No. 5,861,398);etc.

Also of interest are cyclic stresscopin analogs (see U.S. Pat. No.5,663,292). Certain cyclic analogs, e.g. of CRH, have been found to actas antagonists, and have substantially no residual agonist activity.These peptides may have a cyclizing bond initiating, e.g. at theresidues in the 32-position and may optionally have a second such bondinitiating, e.g. at the residues in the 19- or the 20-positions. Eitheror both of these bonds may be an amide bond (or lactam bridge) betweenside chain carboxyl and amino groups. Alternative antagonists includefragments of the stresscopin sequence, as previously described.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of stress related disorders, etc. The compounds may also beused to enhance stresscopin function in weight reduction, treatment ofheart disease, reduction of edema, suppression of anxiety, stressreduction following major surgery, etc. The inhibitory agents may beadministered in a variety of ways, orally, topically, parenterally e.g.subcutaneously, intraperitoneally, by viral infection, intravascularly,etc. Depending upon the manner of introduction, the compounds may beformulated in a variety of ways. The concentration of therapeuticallyactive compound in the formulation may vary from about 0.1-10 wt %.

Antibodies Specific for Stresscopin Polypeptides

The present invention provides antibodies specific for stresscopinpolypeptides, e.g. any one of the variants, polypeptides, or domainsdescribed above. Such antibodies are useful, for example, in methods ofdetecting the presence of stresscopin in a biological sample, and inmethods of isolating stresscopin from a biological sample.

The stresscopin polypeptides of the invention are useful for theproduction of antibodies, where short fragments provide for antibodiesspecific for the particular polypeptide, and larger fragments or theentire protein allow for the production of antibodies over the surfaceof the polypeptide. As used herein, the term “antibodies” includesantibodies of any isotype, fragments of antibodies which retain specificbinding to antigen, including, but not limited to, Fab, Fv, scFv, and Fdfragments, chimeric antibodies, humanized antibodies, single-chainantibodies, and fusion proteins comprising an antigen-binding portion ofan antibody and a non-antibody protein. The antibodies may be detectablylabeled, e.g., with a radioisotope, an enzyme that generates adetectable product, a green fluorescent protein, and the like. Theantibodies may be further conjugated to other moieties, such as membersof specific binding pairs, e.g., biotin (member of biotin-avidinspecific binding pair), and the like. The antibodies may also be boundto a solid support, including, but not limited to, polystyrene plates orbeads, and the like.

“Antibody specificity”, in the context of antibody-antigen interactions,is a term well understood in the art, and indicates that a givenantibody binds to a given antigen, wherein the binding can be inhibitedby that antigen or an epitope thereof which is recognized by theantibody, and does not substantially bind to unrelated antigens. Methodsof determining specific antibody binding are well known to those skilledin the art, and can be used to determine the specificity of antibodiesof the invention for a stresscopin polypeptide, particularly a humanstresscopin polypeptide.

Antibodies are prepared in accordance with conventional ways, where theexpressed polypeptide or protein is used as an immunogen, by itself orconjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, otherviral or eukaryotic proteins, or the like. Various adjuvants may beemployed, with a series of injections, as appropriate. For monoclonalantibodies, after one or more booster injections, the spleen isisolated, the lymphocytes immortalized by cell fusion, and then screenedfor high affinity antibody binding. The immortalized cells, i.e.hybridomas, producing the desired antibodies may then be expanded. Forfurther description, see Monoclonal Antibodies: A Laboratory Manual,Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y., 1988. If desired, the mRNA encoding the heavy and lightchains may be isolated and mutagenized by cloning in E. coli, and theheavy and light chains mixed to further enhance the affinity of theantibody. Alternatives to in vivo immunization as a method of raisingantibodies include binding to phage display libraries, usually inconjunction with in vitro affinity maturation.

Uses of Stresscopin

In light of the pharmacologic activities of stresscopin, numerousclinical indications are evident. For example, clinical indications forwhich a stresscopin peptide or variants thereof may find use includetreatment of obesity, reduction of edema; as an anti-inflammatory agent,as a cardioprotective agent, as a hypotensive agent, as astress-reducing agent, and in the treatment of psychiatric andanxiolytic disorders.

Human obesity is a widespread and serious disorder, affecting a highpercentage of the adult population in developed countries. In spite ofan association with heart disease, type II diabetes, cancer, and otherconditions, few persons are able to permanently achieve significantweight loss. The subject peptides are administered to obese patients forpurposes of appetite suppression. Patients may use various criteria fordetermining obesity. Conveniently, a body mass index (BMI) iscalculated, where a person having a BMI greater than 25 is overweightand may considered for treatment with the subject peptides. Stresscopinsfind use in promoting gastric stasis and anorexic behavior withoutconcomitant activation of the ACTH-glucocorticoid axis.

In a related embodiment, the treatment of non-insulin-dependent diabetesmellitus (NIDDM) is closely related to the treatment of obesity. NIDDMis a metabolic disease that affects about 5% to 7% of the population inwestern countries (and 10% of individuals over age 70). It ischaracterized by hyperglycemia and often accompanied by a number ofother conditions, including hypertension, obesity and lipiddisturbances. Patients are generally categorized as diabetic orhyperglycemic by measuring the level of glucose in the blood, eitherdirectly or by monitoring the level of glycosylated hemoglobin.Treatment is recommended where fasting glucose levels are greater 140mg/dl, where bedtime glucose is greater than 160 mg/dl, or whereHbA_(1c) is greater than 8%. The level of reduction that is desirabledepends on the condition of the patient, and the blood glucose levels atthe start of treatment, but generally about a 10 to 40% reduction isblood glucose is desirable, usually about a 25 to 35% reduction.

The effects of stresscopins on stress related disorders provides a meansof treating affective and mood disorders, which are a group of mentaldisorders characterized by neuroendocrine dysregulation and arecharacterized by a disturbance in the regulation of mood, behavior, andaffect. Affective and mood disorders can have serious impact on anindividual's functional ability, interpersonal relationships andbehavior. Neuroendocrine dysregulation, specifically changes in thehypothalamic-pituitary-adrenal (HPA) axis, has been investigated as abiological correlate of depression. Overall, the HPA axis regulatesphysiologic responses to stress. The hypothalamus controls endocrinefunctions and the autonomic nervous system. It is involved in behaviorsrelated to fight, flight, feeding and mating, many of which are alteredduring episodes of depression.

The hypothalamus releases CRH and related peptides in response tostress, which then stimulates the anterior pituitary to secreteadrenocorticotrophichormone (ACTH). ACTH prompts the adrenal cortex torelease cortisol which, through elaborate feedback mechanisms signalsthe hypothalamus to increase or decrease CRH production. Under ordinarycircumstances, activation of hypothalamic CRH is terminated rapidly bythe negative feedback of rising glucocorticoid levels. However, inmelancholic depression, hypercortisolism does not adequately restrainthe production of CRH in the hypothalamus. Thus, in melancholicdepression, CRH levels are chronically elevated causing hyperactivity ofthe HPA axis. Through administration of stresscopins, the excessiverelease of ACTH is avoided.

Major depression is a syndromal, episodic and recurrent illness withboth psychological and biological components. A diagnosis of bipolardisorder is given to those patients with recurring depression and mania.Those patients with recurrent depression alone have a unipolar pattern.Within the spectrum of depressive illness, there are two distinctsubtypes: melancholic depression and atypical depression. Melancholicdepression is equally common among those with a pattern of unipolar andbipolar depression. Melancholic depression is characterized byhyposomnia (early morning awakening), anorexia and diurnal variation inmood, and is associated with a state of hyperarousal. Atypicaldepression is more common in bipolar patients than in unipolar depressedpatients. Atypical depression is characterized by a state which seems tobe opposite to that of melancholic depression. Patients with atypicaldepression have a syndrome of hypoarousal with hypersomnia, hyperphagia,weight gain and mood liability.

Dysthymia is a chronic disorder characterized by symptoms that includepoor appetite or overeating, low energy (decreased arousal), insomnia orhypersomnia, and poor concentration. These functions are modulated byneuropeptides in the brain, such as CRH and stresscopins. Generally,dysthymia is characterized by hypothalamic CRH levels that are higherthan normal, thereby causing hyperactivity of the HPA axis. However, indysthymia, hypothalamic CRH levels can be lower than normal, causinghypoactivity of the HPA axis, in individuals with a higher than normalbody mass index (BMI). Thus, in dysthymia, hypothalamic CRH levels areinversely related to the BMI of the individual.

Affective disorders are extremely common in general medical practice, aswell as in psychiatry. The severity of these conditions covers anextraordinarily broad range, from normal grief reactions to severe,incapacitating, and sometimes fatal psychosis. Typically these disordersare treated with antidepressant agents or lithium salts. Nevertheless,many shortcomings and problems continue to be associated with all drugsused to treat affective disorders. In addition to less than-dramaticefficacy in some cases, virtually all the drugs used to treat disordersof mood are potentially lethal when acute over dosage occurs and cancause appreciable morbidity even with careful clinical use. Stresscopinsfind use as an anxiolytic agent.

Hypertension is a disease which, if untreated, strongly predisposes toatherosclerotic cardiovascular disease. It is estimated that as many as1 in 4 adult Americans have hypertension. Hypertension is approximatelytwice as common in persons with diabetes as in those without. Theprevalence of hypertension increases with age.

Hypertension should not be diagnosed on the basis of a singlemeasurement. Initial elevated readings should be confirmed on at leasttwo subsequent visits over one week or more with average diastolic bloodpressure of 90 mmHg or greater or systolic blood pressure of 140 mmHg orgreater required for diagnosis of hypertension. Special care iswarranted in diagnosing hypertension in persons with diabetes because ofgreater variability of blood pressure and a much greater likelihood ofisolated systolic hypertension. A goal blood pressure of less than130/85 mmHg is recommended for these patients.

In addition to dietary changes, pharmacological treatment may berequired to control high blood pressure. The subject peptides may beadministered to reduce arterial blood pressure. In addition, a secondaryeffect of reducing hypertension is reduction of edema and inflammatoryexudate volume.

After substantial stress, e.g. major surgery, severe burn, emotionaltrauma, organ transplantation, and other life threatening situations,the subject peptides may be administered to enhance the stress copingresponses. The regulation of the hypothalamo-pituitary-adrenal (HPA)axis in the operative and perioperative period of major surgicalprocedures is necessary for successful adaption to surgical stress. Forexample, plasma ACTH has been found to be highly elevated during asurgical procedure; which were temporally related to CRH levels. Theimmediate postoperative period may be associated with profoundelevations of plasma ACTH, cortisol, and epinephrine. Results haveindicated an altered regulation of the HPA axis in the postoperativeperiod of patients after surgery, which are compatible with similarresults in patients after major abdominal surgery, burned patients, andcritically ill patients.

Pharmaceutical compositions containing stresscopin peptides andderivatives therefrom are useful as cardioprotective agents, e.g. toameliorate ischemic injury or myocardial infarct size consequent tomyocardial ischemia. The development of new therapeutic agents capableof limiting the extent of myocardial injury, i.e., the extent ofmyocardial infarction, following acute myocardial ischemia is a majorconcern of modern cardiology. There has also been interest in thedevelopment of therapies capable of providing additional myocardialprotection which could be administered in conjunction with thrombolytictherapy, or alone, since retrospective epidemiological studies haveshown that mortality during the first few years following infarctionappears to be related to original infarct size.

Myocardial ischemia is the result of an imbalance of myocardial oxygensupply and demand and includes exertional and vasospastic myocardialdysfunction. Exertional ischemia is generally ascribed to the presenceof critical atherosclerotic stenosis involving large coronary arteriesresulting in a reduction in subendocardial flow. Vasospastic ischemia isassociated with a spasm of focal variety, whose onset is not associatedwith exertion or stress. The spasm is better defined as an abruptincrease in vascular tone.

The compounds of this invention can be normally administered orally orparenterally, in the treatment of patients in need of cardioprotectivetherapy. The dosage regimen is that which insures maximum therapeuticresponse until improvement is obtained and thereafter the minimumeffective level which gives relief. Thus, in general, the dosages arethose that are therapeutically effective in producing a cardioprotectiveeffect, i.e., amelioration of ischemic injury or myocardial infarct sizeconsequent to myocardial ischemia. It is also anticipated that thepeptides would be useful as an injectable dosage form which may beadministered in an emergency to a patient suffering from myocardialischemia, etc.

The prevention or inhibition of illness leading to inflammation is ofsignificant concern, particularly for those afflicted with autoimmunediseases such as arthritis and different injuries, includingsports-related injuries and musculoskeletal ailments. Pain usuallyaccompanies inflammation and vice versa. Inflammation involves capillarydilation, with accumulation of fluid and migration of phagocyticleukocytes, such as granulocytes and monocytes, to the site of injury orlesion. Inflammation is important in defending a host against a varietyof infections, but can also have undesirable consequences ininflammatory disorders. Inflammatory conditions include autoimmunediseases; inflammation caused by bacterial and viral infection,including response to vaccination; local inflammation in response totrauma; graft rejection; graft v. host disease, and the like.Stresscopin also finds use in the treatment of different skin diseases.

Conditions of interest for treatment with the subject peptides includemusculoskeletal conditions, both inflammatory and non-inflammatory innature, and acute, subacute or chronic presentation. For example, thecomposition may be used in the treatment of both the early and latestages of inflammatory arthritis, as well as non-infectious inflammatoryarthropathy such as rheumatoid arthritis, bursitis, tendinitis, softtissue injuries, Sjogren's syndrome, systemic lupus erythematous,psoriatic arthritis, gout and other crystalline arthropathies,capsulitis, carpal tunnel syndrome, myositis, polymyalgia, rheumatica,synovitis and Reiter's syndrome. The compositions of this invention mayalso be used in the prevention or treatment of erosive osteoarthritis.Acute and chronic pain and inflammation are often treated withanti-inflammatory/analgesic compounds such as aspirin, ibuprofen andnaproxen. The subject peptides may find use in combinations with thesecompounds.

The stresscopin peptides and derivatives therefrom also find use in thereduction of edema, for example in rheumatoid arthritis, edema secondaryto brain tumors or irradiation for cancer, edema resulting from stroke,head trauma or spinal cord injury, post-surgical edema, asthma and otherrespiratory diseases and cystoid macular edema of the eye.

Formulations

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration. Particularly, agents thatmodulate stresscopin activity, or stresscopin polypeptides and analogsthereof are formulated for administration to patients for the treatmentof stresscopin dysfunction, where the stresscopin activity isundesirably high or low. More particularly, the compounds of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants, gels,microspheres, and aerosols. As such, administration of the compounds canbe achieved in various ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. The stresscopin may be systemic after administration ormay be localized by the use of an implant that acts to retain the activedose at the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination withother pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant is placed in proximity to the site of infection,so that the local concentration of active agent is increased relative tothe rest of the body.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 μg to 100milligrams per kg weight of subject per administration. A typical dosagemay be one tablet taken from two to six times daily, or one time-releasecapsule or tablet taken once a day and containing a proportionallyhigher content of active ingredient. The time-release effect may beobtained by capsule materials that dissolve at different pH values, bycapsules that release slowly by osmotic pressure, or by any other knownmeans of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

The use of liposomes as a delivery vehicle is one method of interest.The liposomes fuse with the cells of the target site and deliver thecontents of the lumen intracellularly. The liposomes are maintained incontact with the cells for sufficient time for fusion, using variousmeans to maintain contact, such as isolation, binding agents, and thelike. In one aspect of the invention, liposomes are designed to beaerosolized for pulmonary administration. Liposomes may be prepared withpurified proteins or peptides that mediate fusion of membranes, such asSendai virus or influenza virus, etc. The lipids may be any usefulcombination of known liposome forming lipids, including cationic lipids,such as phosphatidylcholine. The remaining lipid will normally beneutral lipids, such as cholesterol, phosphatidyl serine, phosphatidylglycerol, and the like.

For preparing the liposomes, the procedure described by Kato et al.(1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids andlumen composition containing the nucleic acids are combined in anappropriate aqueous medium, conveniently a saline medium where the totalsolids will be in the range of about 1-10 weight percent. After intenseagitation for short periods of time, from about 5-60 sec., the tube isplaced in a warm water bath, from about 25-40° C. and this cycle isrepeated about 5-10 times. The composition is then sonicated for aconvenient period of time, generally from about 1-10 sec. and may befurther agitated by vortexing. The volume is then expanded by addingaqueous medium, generally increasing the volume by about from 1-2 fold,followed by shaking and cooling. This method allows for theincorporation into the lumen of high molecular weight molecules.

For use in the above described formulations, stresscopin or derivativestherefrom may be synthesized and stored as a solid lyophilized powderwhich is reconstituted into a pharmaceutically acceptable liquidimmediately prior to use. Such formulations are usually preferredbecause it is recognized by those skilled in the art that lyophilizedpreparations generally maintain pharmaceutical activity better over timethan their liquid counterparts.

In addition, stresscopins and their analogs could be applied topicallyon the skin as well as administered as aerosal sprays.

Alternatively, the peptides may be formulated as a liquid, e.g.comprising a buffer at a concentration of from about 1 mM to about 50 mMthat functions to maintain the pH, wherein the anion of said buffer maybe selected from the group consisting of acetate, phosphate, carbonate,succinate, citrate, borate, tartrate, fumarate and lactate; and analcohol which may be selected from the group consisting of mannitol,sorbitol, ribotol, arabitol, xylitol, inositol, galactitol, methanol,ethanol and glycerol. Other additives may include amino acids such asmethionine, arginine, lysine, glutamic acid, cysteine, glutathione, andthe like, where amino acids are generally present in concentrationsranging from about 1 mM to about 100 mM. Various sugars are optionallyincluded in the formulations, including, for example, glucose, sucrose,lactose, fructose, trehalose, mannose, and the like. Additive sugars aregenerally present in concentrations ranging from about 1% to about 10%.

Stresscopin Nucleic Acids

The invention includes nucleic acids having a sequence set forth in SEQID NO:1 and SEQ ID NO:4; nucleic acids that hybridize under stringentconditions, particularly conditions of high stringency, to the sequencesset forth in SEQ ID NO:1 and SEQ ID NO:2; genes corresponding to theprovided nucleic acids; sequences encoding stresscopins; and fragmentsand derivatives thereof. Other nucleic acid compositions contemplated byand within the scope of the present invention will be readily apparentto one of ordinary skill in the art when provided with the disclosurehere.

The nucleic acids of the invention include nucleic acids having sequencesimilarity or sequence identity to SEQ ID NO:1 and SEQ ID NO:4. Nucleicacids having sequence similarity are detected by hybridization under lowstringency conditions, for example, at 50° C. and 10×SSC (0.9 Msaline/0.09 M sodium citrate) and remain bound when subjected to washingat 55° C. in 1×SSC. Sequence identity can be determined by hybridizationunder stringent conditions, for example, at 50° C. or higher and 0.1×SSC(9 mM saline/0.9 mM sodium citrate). Hybridization methods andconditions are well known in the art, see, e.g., U.S. Pat. No.5,707,829. Nucleic acids that are substantially identical to theprovided nucleic acid sequence, e.g. allelic variants, geneticallyaltered versions of the gene, etc., bind to SEQ ID NO:1 or SEQ ID NO:4under stringent hybridization conditions. By using probes, particularlylabeled probes of DNA sequences, one can isolate homologous or relatedgenes. The source of homologous genes can be any species, e.g. primatespecies, particularly human; rodents, such as rats and mice; canines,felines, bovines, ovines, equines, fish, yeast, nematodes, etc.

In one embodiment, hybridization is performed using at least 18contiguous nucleotides (nt) of SEQ ID NO:1 and SEQ ID NO:4, or a DNAencoding the polypeptide of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQID NO:6. Such a probe will preferentially hybridize with a nucleic acidcomprising the complementary sequence, allowing the identification andretrieval of the nucleic acids that uniquely hybridize to the selectedprobe. Probes of more than 18 nt can be used, e.g., probes of from about18 nt to about 25, 50, 100, 250, or 500 nt, but 18 nt usually representssufficient sequence for unique identification.

Nucleic acids of the invention also include naturally occurring variantsof the nucleotide sequences (e.g., degenerate variants, allelicvariants, etc.). Variants of the nucleic acids of the invention areidentified by hybridization of putative variants with nucleotidesequences disclosed herein, preferably by hybridization under stringentconditions. For example, by using appropriate wash conditions, variantsof the nucleic acids of the invention can be identified where theallelic variant exhibits at most about 25-30% base pair (bp) mismatchesrelative to the selected nucleic acid probe. In general, allelicvariants contain 15-25% bp mismatches, and can contain as little as even5-15%, or 2-5%, or 1-2% bp mismatches, as well as a single by mismatch.

The invention also encompasses homologs corresponding to the nucleicacids of SEQ ID NO:1 and SEQ ID NO:4, or a DNA encoding the polypeptideof SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:6, where thesource of homologous genes can be any mammalian species, e.g., primatespecies, particularly human; rodents, such as rats; canines, felines,bovines, ovines, equines, fish, yeast, nematodes, etc. Between mammalianspecies, e.g., human and mouse, homologs generally have substantialsequence similarity, e.g., at least 75% sequence identity, usually atleast 90%, more usually at least 95% between nucleotide sequences.Sequence similarity is calculated based on a reference sequence, whichmay be a subset of a larger sequence, such as a conserved motif, codingregion, flanking region, etc. A reference sequence will usually be atleast about 18 contiguous nt long, more usually at least about 30 ntlong, and may extend to the complete sequence that is being compared.Algorithms for sequence analysis are known in the art, such as gappedBLAST, described in Altschul et al. Nucl. Acids Res. (1997)25:3389-3402.

The subject nucleic acids can be cDNAs or genomic DNAs, as well asfragments thereof, particularly fragments that encode a biologicallyactive polypeptide and/or are useful in the methods disclosed herein(e.g., in diagnosis, as a unique identifier of a differentiallyexpressed gene of interest, etc.). The term “cDNA” as used herein isintended to include all nucleic acids that share the arrangement ofsequence elements found in native mature mRNA species, where sequenceelements are exons and 3′ and 5′ non-coding regions. Normally mRNAspecies have contiguous exons, with the intervening introns, whenpresent, being removed by nuclear RNA splicing, to create a continuousopen reading frame encoding a polypeptide of the invention.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It can further include the 3′ and 5′untranslated regions found in the mature mRNA. It can further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ and 3′ end of the transcribedregion. The genomic DNA can be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ and 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue, stage-specific, or disease-state specificexpression.

The nucleic acid compositions of the subject invention can encode all ora part of the subject polypeptides. Double or single stranded fragmentscan be obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. Isolated nucleic acids andnucleic acid fragments of the invention comprise at least about 18,about 50, about 100, to about 500 contiguous nt selected from thenucleic acid sequence as shown in SEQ ID NO:1 and SEQ ID NO:4. For themost part, fragments will be of at least 18 nt, usually at least 25 nt,and up to at least about 50 contiguous nt in length or more.

Probes specific to the nucleic acid of the invention can be generatedusing the nucleic acid sequence disclosed in SEQ ID NO:1 and SEQ IDNO:4, or a DNA encoding the polypeptide of SEQ ID NO:2, SEQ ID NO:3, SEQID NO:5 or SEQ ID NO:6. The probes are preferably at least about 18 nt,25nt or more of the corresponding contiguous sequence. The probes can besynthesized chemically or can be generated from longer nucleic acidsusing restriction enzymes. The probes can be labeled, for example, witha radioactive, biotinylated, or fluorescent tag. Preferably, probes aredesigned based upon an identifying sequence of one of the providedsequences. More preferably, probes are designed based on a contiguoussequence of one of the subject nucleic acids that remain unmaskedfollowing application of a masking program for masking low complexity(e.g., BLASTX) to the sequence, i.e., one would select an unmaskedregion, as indicated by the nucleic acids outside the poly-n stretchesof the masked sequence produced by the masking program.

The nucleic acids of the subject invention are isolated and obtained insubstantial purity, generally as other than an intact chromosome.Usually, the nucleic acids, either as DNA or RNA, will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant,” e.g., flanked by one or more nucleotideswith which it is not normally associated on a naturally occurringchromosome.

The nucleic acids of the invention can be provided as a linear moleculeor within a circular molecule, and can be provided within autonomouslyreplicating molecules (vectors) or within molecules without replicationsequences. Expression of the nucleic acids can be regulated by their ownor by other regulatory sequences known in the art. The nucleic acids ofthe invention can be introduced into suitable host cells using a varietyof techniques available in the art, such as transferrinpolycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

Modulation of Stresscopin Expression

The stresscopin genes, gene fragments, or the encoded protein or proteinfragments are useful in gene therapy to treat disorders associated withstresscopin defects. From a therapeutic point of view, inhibitingstresscopin activity has a therapeutic effect on a number of disordersrelating to stress. Inhibition is achieved in a number of ways.Antisense stresscopin sequences may be administered to inhibitexpression. Competitive binding antagonists, for example, a peptide thatmimics stresscopin binding may be used to inhibit activity. Otherinhibitors are identified by screening for biological activity in astresscopin-based binding assay.

Expression vectors may be used to introduce the stresscopin gene into acell. Such vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences. Transcription cassettes may be prepared comprising atranscription initiation region, the target gene or fragment thereof,and a transcriptional termination region. The transcription cassettesmay be introduced into a variety of vectors, e.g. plasmid; retrovirus,e.g. lentivirus; adenovirus; and the like, where the vectors are able totransiently or stably be maintained in the cells, usually for a periodof at least about one day, more usually for a period of at least aboutseveral days to several weeks.

The gene or stresscopin peptide may be introduced into tissues or hostcells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992)Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992) Nature 356:152-154), where gold microprojectiles arecoated with the stresscopin or DNA, then bombarded into skin cells.

Antisense molecules can be used to down-regulate expression ofstresscopin in cells. The anti-sense reagent may be antisenseoligonucleotides (ODN), particularly synthetic ODN having chemicalmodifications from native nucleic acids, or nucleic acid constructs thatexpress such anti-sense molecules as RNA. The antisense sequence iscomplementary to the mRNA of the targeted gene, and inhibits expressionof the targeted gene products. Antisense molecules inhibit geneexpression through various mechanisms, e.g. by reducing the amount ofmRNA available for translation, through activation of RNAse H, or sterichindrance. One or a combination of antisense molecules may beadministered, where a combination may comprise multiple differentsequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996) Nature Biotechnol. 14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in vitro or in an animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see, Wagner et al. (1993), supra and Milligan et al.,supra). Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature which alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 95/23225, and Beigelmanet al. (1995) Nucl. Acids Res. 23:4434-4442). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995) Appl. Biochem. Biotechnol. 54:43-56.

Agents that block stresscopin activity provide a point of interventionin an important signaling pathway. Numerous agents are useful inreducing stresscopin activity, including agents that directly modulatestresscopin expression as described above, e.g. expression vectors,anti-sense specific for stresscopin; and agents that act on thestresscopin protein, e.g. stresscopin specific antibodies and analogsthereof, small organic molecules that block stresscopin bindingactivity, etc.

Diagnostic Uses

DNA-based reagents derived from the sequence of stresscopins, e.g. PCRprimers, oligonucleotide or cDNA probes, as well as antibodies againststresscopins, are used to screen patient samples, e.g. biopsy-derivedtissues, blood samples, etc., for amplified stresscopin DNA, orincreased expression of stresscopin mRNA or proteins. DNA-based reagentsare also designed for evaluation of chromosomal loci implicated incertain diseases e.g. for use in loss-of-heterozygosity (LOH) studies,or design of primers based on stresscopin coding sequence.

The polynucleotides of the invention can be used to detect differencesin expression levels between two samples. A difference between theprotein levels, or the mRNA in the two tissues that are compared, forexample, in molecular weight, amino acid or nucleotide sequence, orrelative abundance, indicates a change in the gene, or a gene whichregulates it, in the tissue of the human that was suspected of beingdiseased.

The subject nucleic acid and/or polypeptide compositions may be used toanalyze a patient sample for the presence of polymorphisms associatedwith a disease state or genetic predisposition to a disease state.Biochemical studies may be performed to determine whether a sequencepolymorphism in a stresscopin coding region or control regions isassociated with disease, particularly stress related disorders, e.g.anxiety disorders. Disease associated polymorphisms may include deletionor truncation of the gene, mutations that alter expression level, thataffect the binding activity of the protein, the kinase activity domain,etc.

Changes in the promoter or enhancer sequence that may affect expressionlevels of stresscopin can be compared to expression levels of the normalallele by various methods known in the art. Methods for determiningpromoter or enhancer strength include quantitation of the expressednatural protein; insertion of the variant control element into a vectorwith a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence, e.g. a disease associated polymorphism.Where large amounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. Cells that expressstresscopin may be used as a source of mRNA, which may be assayeddirectly or reverse transcribed into cDNA for analysis. The nucleic acidmay be amplified by conventional techniques, such as the polymerasechain reaction (PCR), to provide sufficient amounts for analysis. Theuse of the polymerase chain reaction is described in Saiki et al. (1985)Science 239:487, and a review of techniques may be found in Sambrook, etal. Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.14.2-14.33.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin,allophycocyanin,6-carboxyfluorescein(6-FAM),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein(JOE), 6-carboxy-X-rhodamine (ROX),6-carboxy-2,4,7,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g., amplified or cloned fragment, is analyzedby one of a number of methods known in the art. The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to a wild-type stresscopin sequence. Hybridization with thevariant sequence may also be used to determine its presence, by Southernblots, dot blots, etc. The hybridization pattern of a control andvariant sequence to an array of oligonucleotide probes immobilized on anarray, may also be used as a means of detecting the presence of variantsequences. Single strand conformational polymorphism (SSCP) analysis,denaturing gradient gel electrophoresis (DGGE), and heteroduplexanalysis in gel matrices are used to detect conformational changescreated by DNA sequence variation as alterations in electrophoreticmobility. Alternatively, where a polymorphism creates or destroys arecognition site for a restriction endonuclease, the sample is digestedwith that endonuclease, and the products size fractionated to determinewhether the fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels.

Screening for mutations in stresscopins may be based on the functionalor antigenic characteristics of the protein. Protein truncation assaysare useful in detecting deletions that may affect the biologicalactivity of the protein. Various immunoassays designed to detectpolymorphisms in stresscopin proteins may be used in screening. Wheremany diverse genetic mutations lead to a particular disease phenotype,functional protein assays have proven to be effective screening tools.The activity of the encoded stresscopin protein in binding assays, etc.,may be determined by comparison with the wild-type protein. Proteins mayalso be screened for the presence of post-translational modification ofthe stresscopin proteins, e.g. under pathological conditions, includingproteolytic fragments, amidation, acetylation etc.

Antibodies specific for stresscopin may be used in staining or inimmunoassays. Samples, as used herein, include biological fluids such asblood, cerebrospinal fluid, dialysis fluid and the like; organ or tissueculture derived fluids; and fluids extracted from physiological tissues.Also included in the term are derivatives and fractions of such fluids.The cells may be dissociated, in the case of solid tissues, or tissuesections may be analyzed. Alternatively a lysate of the cells may beprepared.

Diagnosis may be performed by a number of methods to determine theabsence or presence or altered amounts of normal or abnormal stresscopinin patient cells. For example, detection may utilize staining of cellsor histological sections, performed in accordance with conventionalmethods. Cells are permeabilized to stain cytoplasmic molecules. Theantibodies of interest are added to the cell sample, and incubated for aperiod of time sufficient to allow binding to the epitope, usually atleast about 10 minutes. The antibody may be labeled with radioisotopes,enzymes, fluorescers, chemiluminescers, or other labels for directdetection. Alternatively, a second stage antibody or reagent is used toamplify the signal. Such reagents are well known in the art. Forexample, the primary antibody may be conjugated to biotin, withhorseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to afluorescent compound, e.g. fluorescein rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

In some embodiments, the methods are adapted for use in vivo. In theseembodiments, a detectably-labeled moiety, e.g., an antibody, which isspecific for stresscopin is administered to an individual (e.g., byinjection), and labeled cells are located using standard imagingtechniques, including, but not limited to, magnetic resonance imaging,computed tomography scanning, and the like.

Diagnostic screening may also be performed for polymorphisms that aregenetically linked to a disease predisposition, particularly through theuse of microsatellite markers or single nucleotide polymorphisms.Frequently the microsatellite polymorphism itself is not phenotypicallyexpressed, but is linked to sequences that result in a diseasepredisposition. However, in some cases the microsatellite sequenceitself may affect gene expression.

Microsatellite linkage analysis may be performed alone, or incombination with direct detection of polymorphisms, as described above.The use of microsatellite markers for genotyping is well documented. Forexamples, see Mansfield et al. (1994) Genomics 24:225-233; Ziegle et al.(1992) Genomics 14:1026-1031; Dib et al., supra.

Diagnostic screening may also be performed for polymorphisms that aregenetically linked to a predisposing mutation, particularly through theuse of microsatellite markers or single nucleotide polymorphisms.Frequently the microsatellite polymorphism itself is not phenotypicallyexpressed, but is linked to sequences that result in a diseasepredisposition. However, in some cases the microsatellite sequenceitself may affect gene expression. Microsatellite linkage analysis maybe performed alone, or in combination with direct detection ofpolymorphisms, as described above. The use of microsatellite markers forgenotyping is well documented. For examples, see Mansfield et al. (1994)Genomics 24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031; Dib etal., supra.

The detection methods can be provided as part of a kit. Thus, theinvention further provides kits for detecting the presence of an mRNAencoding stresscopin, and/or a polypeptide encoded thereby, in abiological sample. Procedures using these kits may be performed byclinical laboratories, experimental laboratories, medical practitioners,or private individuals. The kits of the invention for detecting apolypeptide comprise a moiety that specifically binds the polypeptide,which may be a specific antibody. The kits of the invention fordetecting a nucleic acid comprise a moiety that specifically hybridizesto such a nucleic acid. The kit may optionally provide additionalcomponents that are useful in the procedure, including, but not limitedto, buffers, developing reagents, labels, reacting surfaces, means fordetection, control samples, standards, instructions, and interpretiveinformation.

Genetically Altered Cell or Animal Models for Stresscopin Function

The subject nucleic acids can be used to generate transgenic animals orsite specific gene modifications in cell lines. Transgenic animals maybe made through homologous recombination, where the normal stresscopinlocus is altered. Alternatively, a nucleic acid construct is randomlyintegrated into the genome. Vectors for stable integration includeplasmids, retroviruses and other animal viruses, YACs, and the like.

The modified cells or animals are useful in the study of stresscopinfunction and regulation. For example, a series of small deletions and/orsubstitutions may be made in the stresscopin gene to determine the roleof different residues in receptor binding, signal transduction, etc. Ofinterest is the use of stresscopin to construct transgenic animal modelsfor stress related disorders, where expression of stresscopin isspecifically reduced or absent. Specific constructs of interest includeanti-sense stresscopin, which will block stresscopin expression andexpression of dominant negative stresscopin mutations. A detectablemarker, such as lac Z may be introduced into the stresscopin locus,where up-regulation of stresscopin expression will result in an easilydetected change in phenotype.

One may also provide for expression of the stresscopin gene or variantsthereof in cells or tissues where it is not normally expressed or atabnormal times of development. By providing expression of stresscopinprotein in cells in which it is not normally produced, one can inducechanges in cell behavior, e.g. in the control of cell growth andtumorigenesis.

DNA constructs for homologous recombination will comprise at least aportion of the stresscopin gene with the desired genetic modification,and will include regions of homology to the target locus. The regions ofhomology may include coding regions, or may utilize intron and/orgenomic sequence. DNA constructs for random integration need not includeregions of homology to mediate recombination. Conveniently, markers forpositive and negative selection are included. Methods for generatingcells having targeted gene modifications through homologousrecombination are known in the art. For various techniques fortransfecting mammalian cells, see Keown et al. (1990) Methods inEnzymology 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in culture. The transgenic animalsmay be any non-human mammal, such as laboratory animals, domesticanimals, etc. The transgenic animals may be used in functional studies,drug screening, etc., to determine the effect of a candidate drug onstress responses.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Materials and Methods

Cloning, Sequencing and Expression Analysis of Human Stresscopin cDNA.

The identity of stresscopin 1 mRNA was deduced by comparisons ofmultiple human genomic DNA sequences (AC024179 and AC005903) and apartial EST sequence (BE390203). The deduced ORF of stresscopin 1 wasverified by PCR using nested gene-specific primers and Marathon-readycDNA templates (Clontech, Inc., Palo Alto, Calif.) from human testis andprostate. Stresscopin 2 was initially identified as a partial cDNA(AW293249) from a human subtracted library NCI_CGAP_Sub4. The identityof this gene was verified by rapid amplification of 3′ cDNA ends using ahuman Marathon-ready colon cDNA library.

Amplified cDNA fragments were gel-purified and subcloned followingblunt-end ligation. To determine the expression profile of thestresscopin 1 gene, stresscopin transcript in 23 human tissues wasamplified by high stringency PCR using a panel of genomic DNA-free firststrand cDNAs primed with oligo-d(T) primer (Origene Technologies, Inc.,Rockville, Md.) as template.

Immunohistochemical analysis. Specific rabbit anti-stresscopin 1 andanti-stresscopin 2 antibodies were generated using synthetic peptidescorresponding to the mature region of stresscopin 1 and stresscopin 2,respectively, as the antigen (Strategic Biosolutions, Ramona, Calif.).The stresscopin peptides were conjugated to the carrier protein keyholelimpet hemocyanin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride before immunization. Antibodies were purified usingantigen-conjugated affinity columns and their titers determined byELISA.

Tissues were obtained from adult male mice and embedded in paraffinfollowing fixation in Bouin's solution. Following incubation in xylene,tissue sections were blocked with 5% goat serum in PBS to saturatenonspecific binding sites. Sections were then incubated with thespecific anti-stresscopin antibody for 2 h at room temperature in amoist chamber before washing for three times (20 min each) in PBS with0.1% Tween 20. Following incubation with the primary antibody, sectionswere incubated with gold-conjugated goat anti-rabbit IgG. Sections werethen washed before being stained with the SilvEnhance solution (ZymedLaboratory, Inc., South San Francisco, Calif.) and counterstained withhematoxylin. Negative controls were performed by substituting theprimary antibodies with antibodies presaturated with the peptideantigen.

Peptide synthesis and analysis. The stresscopin peptides with greaterthan 95% purity were synthesized using a Symphony/Multiplex™ automatedpeptide synthesizer based on the solid phase fluorenylmethoxycarbonylprotocol. All peptides synthesized were routinely analyzed by reversephase HPLC with a Vydac C18 analytical column and Mass Spectrometryusing a MALDI-TOF (matrix-assisted laser desorption ionization-time offlight) Voyager-DE RP Biospectrometry Workstation. Full-lengthstresscopin prepared with this protocol agrees with a calculated 4691 MWof the amidated form of stresscopin. CRH and urocortin were purchasedfrom Sigma Biochemicals, Inc. (St. Louis, Mo.). Astressin was purchasedfrom Bachem Feinchemicalien, Bubendorf, Switzerland. Radiolabeled [¹²⁵I]human urocortin (2000 Cu/mmole) tracer was purchased from AmershamPharmacia (Arlington Heights, Ill.).

Construction of Expression Vectors for CRH Receptors and the cAMP Assay.

To obtain full-length type 1 and type 11 CRH receptor cDNAs forfunctional assays, nested gene-specific primers flanking the receptorORF were used to PCR full-length cDNA fragments of CRH R1, CRH R2-α, andCRH R2β receptors. The receptor cDNA fragments were subcloned into thepcDNA3.1/Zeo vector with a prolactin signal peptide for secretion andtriple FLAG/M1 epitope cassette upstream of the multiple cloning site.CRH receptor expression vectors were transfected into human 293T cellsunder serum-free conditions for 2h followed by incubation in DMEM/F12medium with 10% fetal bovine serum (FBS) for another 48 h. For theestimation of adenylate cyclase activation by stresscopin and relatedhormones, transfected cells were seeded at a concentration of 100,000cell/well in 48 well culture plates in DMEM/F12 medium containing 0.1%BSA and 2.5mM IBMX for 16 h. Contents of cAMP in whole cell lysate weredetermined using a cAMP radioimmunoassay. To analyze the effects ofstresscopin and related hormones on cAMP accumulation in cellsconstitutively expressing CRH receptors, a human retinoblastoma cellline (Y79) and a rat cardiac cell line (A7r5) were purchased from ATCC(Manassas, Va.) and maintained in DMEM/F12 medium with 20% FBS beforetreatment with different hormones.

Phylogenetic inference. A multiple alignment of selected vertebrate CRHfamily protein sequences was constructed with ClustaIX and correctedusing a published alignment of mature protein data. Phylogeneticanalysis was carried out by the neighboring-joining method as well asthe BlockMaker using Gibbs method.

Receptor Binding assay. Human 293T cells expressing different isoformsof CRH R2 were incubated with 50,000 cpm (0.1 g) of ¹²⁵I-urocortin andvarious concentrations of nonradioactive peptides diluted in a bindingbuffer consisting of PBS, a protease inhibitor cocktail (SigmaBiochemicals, Inc.), and 0.1% BSA. After 60 min incubation at 3TC, thecell-associated ligand was estimated following centrifugation andrepeated washing. Radioactivity of samples was determined in aGENESYS-counter (Laboratory Technologies, Inc., Maple Park, Ill.).

Food intake and body weight in food-restricted mice. Six week-old malemice (20-25 g b.w.) of the inbreed Balb/C strain were housedindividually in a regulated environment with 12L/12D light cycle. Beforefeeding tests, mice were deprived of food for 16h with free access towater and injected with testing reagents i.p. at 10 AM the followingday. Food intake was measured by placing preweighed pellets in the cageand weighing the uneaten pellets at 2, 4 and 8 h after injection. Bodyweight was also monitored at 0, 2, 4, and 8 h.

Effects on gastric emptying in food-restricted mice. To study theeffects on gastric emptying, mice were deprived of food for 16 h withfree access to water. The fasted mice were then given free access topreweighed pellets for 90 min. and were injected i.p. with differenthormones or saline. The mice were deprived of food again after i.p.injection and sacrificed by cervical dislocation at 2 h. afterinjection. The stomach was excised at point of the pylorus and cardiabefore determination of its wet weight. Gastric emptying was calculatedby comparison with stomach weight of control mice sacrificed at 0 h.following injection. See, Asakawa, A. et al. (1999) Gastroenterology116:1287-1292.

Effects on heat-induced paw edema formation in anaesthetized rats. Theanti-inflammatory effect of stresscopin and related peptides wereassayed using an established model (Turnbull et al. (1996) Euro. J.Pharmacol. 303:213-216). Briefly, 5-week-old male

Sprague-Dawley rats were injected with 20 nM of the testing peptide andanaesthetized with ketamine (100 mg/Kg). Thirty min. later, paw edemawas induced following a one minute exposure to hot water at 58° C. Theanimals were sacrificed 30 min later. Both paws were removed at theankle joint and weighed. The degree of edema was estimated as thedifferences in weight gain between the heated and unheated paw dividedby the weight of the unheated paw.

In Vivo and in Vitro assay of pituitary ACTH releasing activity.Anterior pituitaries were obtained from 6-week-old male Sprague-Dawleyrats. Following dispersion using collagenase and mechanical pipetting,cells were resuspended and cultured for 3 days in DMEM with 10% FBS(2×10⁵ cells/well). Before hormonal treatment, cells were washed twicewith serum-free medium followed by incubation with DMEM/F12 mediacontaining 0.1% BSA, 2.5 mM IBMX, and different hormones (1 nM). After 2h at 37° C., media were collected and assayed for ACTH contents using aradioimmunoassay from Diagnostic Systems Laboratories, Inc. (Webster,Tex.). To detect the ACTH-releasing activity of stresscopins and relatedpeptides in vivo, 6-week-old male Sprague-Dawley rats were injected withdifferent hormones i.p. (2 nmoles/kg) and sacrificed 30 min followingtreatment. Whole blood was collected with 60 IU/ml heparin and serumobtained for ACTH measurement.

Results

As shown in FIG. 1, there is significant homology between the providedstresscopin sequences, and other genes in the corticitropin releasinghormone gene family. Two human paralogs of CRH/urocortin have beenidentified, stresscopin 1 and stresscopin 2. A putative stresscopinortholog from Japanese pufferfish (Takifugu rubripes) is alsoidentified. Stresscopin 1 encodes a prepro-protein of 112 amino acidsand a putative mature protein of 43 amino acids whereas the161-amino-acid open reading frame (ORF) of stresscopin 2 contains apredicted 41-amino-acid mature peptide (FIGS. 1 a and 1 b).

Although the overall amino acid sequences of stresscopins from human andfish showed no similarity to known proteins, a stretch of 30 residues attheir C-termini adopted an extended α-helical structure shared by allCRH family peptides (FIG. 1 c). Both human stresscopin ORFs contain asignal peptide for secretion. The predicted mature regions are flankedby potential proteolytic cleavage sites and an alpha-amidation donorresidue. The identity of stresscopin 1 and stresscopin 2 transcripts wasconfirmed following PCR of cDNAs from human testis and colon,respectively.

Alignment of the mature peptides with related CRH family hormonesindicated that mature stresscopins from human and pufferfish, but notthe prepro-regions, show 35-38% identity to other family proteins (FIG.1 c). These novel peptides share identical secondary structures,although the predicted structures of mature stresscopin 1 andstresscopin 2 are distinct from that of other family peptides at theirN-terminus (FIG. 1 c). Phylogenetic analysis of nine CRH family proteinsfrom fish, frog, and mammals suggested the ancient evolution of threesubgroups of CRH family proteins, with the human and pufferfishstresscopins clustered in a separate branch (FIG. 1 d).

Based on PCR analysis using a panel of human cDNAs (1 ng/reaction) from23 different tissues, stresscopin 1 transcript was found in brain andmultiple peripheral tissues (heart, kidney, spleen, lung, muscle,stomach, testis, placenta, thyroid, adrenal, pancreas, ovary, peripheralblood cells, bone marrow, and fetal liver) (FIG. 2 a, panel 1). The sameanalysis using diluted template cDNA (10 pg/reaction) shows thestresscopin 1 transcript only in brain, heart, adrenal, and peripheralblood cells, suggesting a relatively greater abundance of stresscopin 1expression in these tissues (FIG. 2 a, panel 2).

Likewise, PCR analysis showed that the stresscopin 2 transcript could bedetected in most tissues analyzed with colon, small intestine, muscle,stomach, thyroid, adrenal, and pancreas showing greater levels ofexpression (FIG. 2 a, panels 3 and 4). Because heart and digestivetissues showed relatively higher expression of the stresscopin 1 andstresscopin 2 transcripts, respectively, a comparative analysis wasperformed for stresscopin 1 mRNA in different cardiac compartments andstresscopin 2 transcript in the digestive system.

As shown in FIGS. 2 b and 2 c, the stresscopin 1 transcript could beamplified in various regions of human heart whereas the stresscopin 2transcript was detected in the ascending colon, descending colon,transverse colon, duodenum, Ileum, jejunum, stomach, but not inIleocecum, cecum, rectum, liver, and esophagus. In addition,immunohistochemical analysis using an anti-stresscopin 1 antibodydetected specific stresscopin 1 signals in different regions of mouseheart with the atrium tissues showing a higher level of expression (FIG.2 d, left panel). In addition, immunoreactive stresscopin 1 was detectedin the posterior pituitary (FIG. 2 e, left panel). In contrast, specificstaining for stresscopin 2 was detected in the muscularis mucosae of thesmall intestine using an anti-stresscopin 2 polyclonal antibody (FIG. 2f, left panel). Negative control staining using antibodies presaturatedwith free antigens showed no specific signals (FIGS. 2 d, 2 e, and 2 f;right panels).

While stresscopin peptides could be the ligand for orphan GPCRs, theobservation that ligand-receptor pairs usually co-evolved in diversevertebrates suggests that the putative receptors for stresscopinpeptides are likely related to the known CRH receptors and other group BGPCRs. Global sequence analysis based on all GPCR sequences in theGenBank using both pairwise sequence comparison and phylogenetic treebuilding indicated that the type-1 and type-2 CRH receptors are the mostlikely candidates to mediate the action of stresscopins because otherclosely related GPCRs are known to bind ligands with highly divergedstructures (e.g. secretin and glucagon-related peptides) whereas noknown orphan GPCR has an intermediate similarity. Two cell linesexpressing CRHR1 (human retinoblastoma cell Y79) or CRHR2 (rat cardiaccell A7r5) were treated with the synthetic stresscopin 1 peptide. Asshown in FIG. 3 a, treatment with stresscopin 1 stimulated cAMPproduction by the cardiac cell line.

Surprisingly, this peptide was ineffective in Y79 cells (FIG. 3 b),suggesting that stresscopin 1 activates only the type-2 CRH receptor. Incontrast, both CRH and urocortin stimulated cAMP production in both celllines. To expand this observation, 293T cells transiently transfectedwith different CRH receptor cDNAs were treated with syntheticstresscopin 1, stresscopin 2, or the pufferfish stresscopin peptide.Analysis of cAMP production showed that stresscopins are potent agonistsfor two isoforms of recombinant CRHR2, but not CRHR1 (FIGS. 3 c-3 e).Again, CRH and urocortin stimulated cAMP production mediated by allthree receptors. While both human stresscopins and the pufferfishortholog selectively activated type-2 CRH receptors, stresscopin 1appeared to have a higher potency (>10-fold) as compared to stresscopin2 and the pufferfish peptide.

Because the proteolytic processing sites flanking the mature stresscopin1 peptide do not correspond to those found in other CRH family peptides,293T cells expressing CRHR1 or CRHR2 were treated with a panel oftruncated stresscopin 1 peptides with deletions of 1 to 5 amino acids atthe N-terminus and a nonamidated stresscopin 1 peptide to test thestructural requirement for the selective activation of type-2 CRHR bystresscopin 1. As shown in FIG. 3 f, full-length stresscopin 1 andtruncated peptides all stimulated cAMP production by recombinant CRHR2,but not CRHR1. However, a stresscopin 1 peptide without amidation at theC-terminus showed a >50-fold reduction in its ability to stimulate cAMPproduction, suggesting that α-amidation is important for generatingbioactive stresscopin 1. Furthermore, radioligand receptor bindingassays using labeled urocortin confirmed the ability of stresscopin 1and 2 peptides to bind CRHR2 and CRHR2 (FIGS. 3 g and 3 h).

To confirm the specific activation of CRHR2 by stresscopin peptides,tests were conducted to determine the ability of human stresscopins andrelated hormones to stimulate ACTH secretion by cultured rat anteriorpituitary cells, and to elicit ACTH release in intact male rats. Asshown in FIGS. 4 a and 4 b, both in vitro and in vivo treatments withCRH and urocortin, but not stresscopin 1 or stresscopin 2, stimulatedthe release of ACTH, which is presumably mediated by CRH R1. Earlierstudies indicated that CRH R2 mutant mice failed to show the enhancedcardiac performance or reduced blood pressure associated with systemicurocortin, but exhibited increased edema formation in response tothermal exposure. Based on the known association betweenurocortin-induced hypotension and anti-edema responses, the effects ofstresscopins on heat-induced edema were tested to determine if edema ismediated by CRH R2. As shown in FIG. 4 c, i.p. administration withstresscopin 1 or stresscopin 2 suppressed heat-induced edema formationin anesthetized rats, similar to that induced by urocortin and CRH.Because CRHR2 is essential for sustained feeding suppression induced byurocortin, the ability of stresscopins to regulate anorexic responseswas also studied based on cumulative food intake in fasting mice.

As shown in FIG. 4 d, i.p. treatment with stresscopin 1 (left panel) orstresscopin 2 (right panel), like CRH and urocortin, dose-dependentlydecreased food intake in fasting mice. In contrast, a truncatedstresscopin 1 peptide with a deletion of the first 10 amino acids at theN-terminus (SCP1(11-43)) has no effect on food intake in mice (leftpanel). Furthermore, stresscopin 1 and stresscopin 2 also suppressedgastric emptying activity as found for urocortin and CRH (FIG. 4 e).This suggests that the anorexic effects of stresscopins are partlymediated at the level of the stomach.

As a result, these tests show stresscopin 1 and stresscopin 2 are novelselective and cogent ligands for CRHR2, and are likely important in themediation of anorexic and vascular responses following stress. UnlikeCRH and urocortin, stresscopins do not elicit ACTH release and theresultant elevations in glucocorticoids.

Initial stress-induced responses, such as gluconeogenesis and increasesin arterial pressure and heart rate, provide a vital short-termmetabolic lift, but prolonged or inappropriate exposure to stress cancompromise homeostasis thereby leading to disease. Because CRHR2 isbelieved to be important in the regulation of the recovery phase of thestress response, the present findings suggest that stresscopin peptidesrepresent important hormones in the protection of the organism to avoiddamage incurred by prolonged and excessive exposure to the initial“flight or fight” response. This response is characterized by theactivation of the CRH/ACTH/glucocorticoid axis and the release ofcatecholamines by the sympathetic adrenomedullary network. Thestress-coping or “countershock” responses mediated by stresscopins inboth central and peripheral tissues likely include the hypotensive,cardioprotective, anxiolytic, and anorexic responses mediated by CRH R2expressed in brain, posterior pituitary, cardiac and skeletal muscle,spleen, and the gastrointestinal tract.

It is clear that adaptive responses induced by stressors are mediated bythe autonomic nervous system and two interrelated and somewhatantagonistic CRH receptor pathways. Although the four mammalianCRH-related peptide hormones, CRH, urocortin, stresscopin 1, andstresscopin 2 show overlapping specificity to CRH R1 and CRH R2, optimalresponses to stress depend on an integrated release of theseendocrine/paracrine ligands in a tissue-specific and time-coordinatedmanner.

Table 1, shown below, describes the effects on body weight change andaccumulative food intake in mice treated with stresscopin 1 orstresscopin 2. At 2 h after treatment, body weight and food intake werereduced in stresscopin-treated animals as compared to control micereceiving saline vehicle.

TABLE 1 Effects of Stresscopin 1 and Stresscopin 2 on food intake andbody weight change in fasted animals. Stresscopin 1 Stresscopin 2Stresscopin 2 Control (20 nM) (2 nM) (20 nM) Average body 0.98 ± 0.20.31 ± 0.14* −0.19 ± 0.24* 0.11 ± 0.1* weight change/mouse (grams, N =5) Average food 0.675 0.185 0.176 0.152 intake/mouse (grams, N = 5)*Significantly different from control animals.

The experiments described herein should not be considered limiting onthe invention, but merely illustrative to one skilled in the art, of thewide spectrum of possibilities for using stresscopin 1 and 2.

What is claimed is:
 1. A method of treating a metabolic diseasecomprising administering to a subject in need thereof, a vectorcomprising a nucleic acid molecule encoding the polypeptide as set forthin SEQ ID NO:5 or 6 or sequences having at least 90% identity to SEQ IDNO:5 or
 6. 2. The method of claim 1, wherein the disease is diabetes orheart disease.
 3. The method of claim 1, wherein the vector is a plasmidor a viral vector.
 4. The method of claim 3, wherein the vector is anadenoviral vector or a retroviral vector.
 5. The method of claim 1,wherein the vector comprises a transcription cassette operably linked tothe nucleic acid sequence encoding the polypeptide of SEQ ID NO:5 or 6.6. The method of claim 1, wherein the subject is a human.